U.S. patent number 3,872,074 [Application Number 05/264,441] was granted by the patent office on 1975-03-18 for method for the aminolysis of activated esters in the presence of n-hydroxy compounds as catalysts.
This patent grant is currently assigned to Farbwerke Hoechst Aktiengesellschaft. Invention is credited to Rolf Geiger, Wolfgang Konig, Hans Wissmann.
United States Patent |
3,872,074 |
Konig , et al. |
March 18, 1975 |
**Please see images for:
( Certificate of Correction ) ** |
Method for the aminolysis of activated esters in the presence of
N-hydroxy compounds as catalysts
Abstract
A process for the manufacture of peptides by reacting a
protected amino acid or a protected peptide ester with an
optionally protected amino acid or a peptide having a free amino
group, which may be bound to a polymer support, by adding a
heterocyclic compound of the formula ##SPC1## Wherein X stands for
the groups C=O, C=S or -N= and X and N are members of a 5- or
6-membered heterocyclic ring optionally substituted and fused with
a benzene nucleus and/or optionally containing 1 or 2 further
hetero atoms, and the pK-value whereof lies between 3.7 and 4.2, as
catalysts for the condensation.
Inventors: |
Konig; Wolfgang
(Langenhaim/Taunus, DT), Geiger; Rolf
(Frankfurt/Main, DT), Wissmann; Hans (Bad
Soden/Taunus, DT) |
Assignee: |
Farbwerke Hoechst
Aktiengesellschaft (Frankfurt/Main, DT)
|
Family
ID: |
5833518 |
Appl.
No.: |
05/264,441 |
Filed: |
June 20, 1972 |
Foreign Application Priority Data
|
|
|
|
|
Jan 20, 1972 [DT] |
|
|
2202613 |
|
Current U.S.
Class: |
530/333; 530/334;
930/10; 530/338 |
Current CPC
Class: |
C07K
5/06043 (20130101); C07K 14/695 (20130101); C07K
14/625 (20130101); C07K 1/10 (20130101); A61K
38/00 (20130101) |
Current International
Class: |
C07K
1/10 (20060101); C07K 1/00 (20060101); C07K
14/435 (20060101); C07K 14/625 (20060101); C07K
14/695 (20060101); C07K 5/06 (20060101); C07K
5/00 (20060101); A61K 38/00 (20060101); C07c
103/52 (); C07g 007/00 () |
Field of
Search: |
;260/112.5 |
Foreign Patent Documents
Other References
Koenig et al., Chem. Ber., 103, 788-798 (1970). .
Koenig et al., Chem. Ber., 103, 2034-2040 (1970). .
Merrifield, Adv. in Enzymology, 32, 252 (1969)..
|
Primary Examiner: Gotts; Lewis
Assistant Examiner: Suyat; Reginald J.
Attorney, Agent or Firm: Curtis, Morris & Safford
Claims
1. In a method for synthesizing a peptide which comprises reacting
a protected amino acid or protected peptide of the formula
##SPC6##
with a protected or unprotected amino acid or peptide of the
formula ##SPC7##
wherein V is an amino protective group or pyroglutamyl: Y is a
protected or unprotected side chain of a natural or synthetic
.alpha.-or .beta.-amino acid, or ##SPC8## may be pyrrolidinyl; R is
di-, tri-, tetra-, or penta-chlorophenyl, nitrophenyl, or
chloronitrophenyl; m.sub.1 is zero or an integer from 1 to about
10; m.sub.2 is zero or an integer from 1 to an order of magnitude
of 500; and W is hydroxy, O-alkyl, O-aralkyl, amino-alkylamino,
aralkylamino, or arylamino, or a polymeric carrier bonded by an
ester or amide bond, the improvement wherein the reagents are
reacted in a strongly polar solvent in the presence of a catalytic
compound of the formula ##SPC9##
wherein X is C=O, C=S, or N= and X and N are members of a 5-or
6-membered substituted or unsubstituted heterocyclic ring, or of
such a ring containing 1 or 2 additional hetero atoms, or of such
rings fused with a substituted or unsubstituted benzene nucleus,
said catalytic compound having a pK-value for the -N=OH group
thereof which is between 3.7 and 4.2, and wherein the substituents
on said substituted heterocyclic ring and on said substituted
benzene nucleus are selected from the group consisting of lower
alkyl, lower alkoxy, halo, perhalo lower alkyl and
2. A method as in claim 1 wherein said pK value is measured at
30.degree.C. in a 0.5 molar solution in a 6:4 solvent mixture of
diethylene glycol dimethyl ether and water.
Description
The present invention relates to a process for the manufacture of
peptides.
In many cases, peptides are prepared with a good yield, using
strongly activated esters of N-acylamino acids, for example
N-hydroxy-succinimide esters [cf. J. Am. Chem. Soc. 86 1839 (1964
)], 1-hydroxy-benzotriazole esters [cf. Chem. Ber. 103, 788 (1970
)], or 1-hydroxy-4-oxo-3,4-dihydrobenzotriazine esters [cf. Chem.
Ber. 103, 2034 (1970)]. These strongly activated esters, however,
cannot always be prepared in crystallized form and with the degree
of purity required. Moreover, they are relatively unstable and
often cannot be stored for a long time. Better crystallization
properties and also a higher stability are inherent in negatively
substituted phenyl esters, for example p-nitrophenyl esters (cf.
Chem. u. Ind. 1955, page 1517), 2,4,5-trichlorophenyl esters [cf.
Helv. Chim. Acta 46, 1609 (1963)], pentachlorophenyl esters [cf.
Roczniki Chem. 35, 1533 (1961)], or 2,6-dichloro-4-nitrophenyl
esters [cf. Aust. J. Chem. 21, 477 (1968)]. However, these esters
have the disadvantage of considerably reducing the reaction rate in
the synthesis of sterically hindered peptides, the result being, in
many cases, a reaction period of many days. In extreme cases, no
peptides are formed at all.
Attempts have already been made for some time to catalyse the
esters which are slow to react by means of additives of various
types. For example, imidazole [cf. Angew. Chem. 74, 904 (1962); op.
cit. 75, 209 (1963)], 2-hydroxy-pyridine, 1,2,4-triazole or
pyrazole derivatives [cf. Proc. Chem. Soc. 1963, page 266; Rec.
Trav. chim. Pays-Bas 84, 213 (1965)] have been used for the
catalysis of esters.
Although said catalysts, except imidazole, caused aminolysis of the
activated esters without racemization, the reaction rate could not
be increased to such an extent that peptides, which are heavily
sterically hindered, are also formed smoothly.
Hence, these catalysts are weak and their activity is even attained
by polar basic solvents, for example dimethylformamide.
Accordingly, they do not further accelerate the reaction rate in
the solvents which are mostly used for the synthesis of higher
peptides.
An improvement was made using the sodium salt of 2-hydroxypyridine
for the catalysis (cf. German Offenlegungsschrift No. 2 031 826).
After scarcely an hour, good yields are obtained even with
sterically hindered peptides. However, the sodium salt of
2-hydroxy-pyridine has the disadvantage of giving a strongly basic
reaction and, possibly, bringing about racemization in the
sensitive peptide molecules. In fact, a racemization test by gas
chromatography showed that the sodium salt of 2-hydroxy-pyridine
has a racemizing effect. It has now been found that aminolysis of
activated esters of the phenyl ester type can be extraordinarily
accelerated by adding an acid heterocyclic N-hydroxy compound
having a pK-value of from 3.7 to 4.2, so that a synthesis of
peptides, which would otherwise take a reaction time of many days,
is complete within a few minutes.
A comparison of the half-life periods in the synthesis of
Z-Val-cyclohexylamide from molar amounts of Z-Val-ONp and
cyclo-hexylamine with the addition of the various hydroxy compounds
with the pK-values of these compounds demonstrates especially well
the catalytic activity of the N-hydroxy compounds as dependent on
the pK-value (cf. Table 1, experimental part).
This invention now provides a process for the manufacture of
peptides, which comprises reacting a protected amino acid or a
protected peptide of the formula II ##SPC2##
in which V stands for an amino protective group usual in peptide
chemistry on the pyroglutamyl radical, Y for a lower alkyl group
which may be branched and/or substituted by hydroxy, amino, thio,
carboxylic acid, carbamoyl, guanidino, aryl, imidazoyl or indolyl
radicals which may be suitably protected, where required, and the
group --NH--CHY-- may also stand for the pyrrolidinyl radical, R
stands for a di-, tri-, tetra- or penta-chlorophenyl, nitrophenyl
or chloronitrophenyl radical and m.sub.1 stands for zero or an
integer of from about 1 to 10, while adding a compound of the
general formula I ##SPC3##
in which X stands for the groups C = O, C = S or --N=, and X and N
are members of a 5- to 6-membered, optionally substituted,
heterocyclic ring which may be fused with a benzene nucleus and/or
which contains 1 or 2 further hetero atoms, and the pK value of
which in a 0.5-molar solution in a mixture of 6 parts of
diethylene-glycol dimethyl ether and 4 parts of water at
30.degree.C is between 3.7 and 4.2, with an amino acid or a peptide
which may be protected and which corresponds to the general formula
III ##SPC4##
in which Y and the --NH--CHY-- radical are defined as above, and
m.sub.2 stands for zero or integers on the order of magnitude of 1
to 500, and W stands for a hydroxy, O-alkyl, O-aralkyl or amino
group which may be substituted by alkyl, aralkyl or aryl, or for a
polymer support linked in ester- or amide-like form and, where
required, all, or part of, the protective groups are split off in
known manner from the so-obtained protected peptide of the formula
IV ##SPC5##
The pK-value of the additives is critical for successfully carrying
out the process of the invention. As disclosed in the Experimental
Part, it is necessary to measure the pK-value. If, in the system
mentioned, the pK-value of the compounds of the formula I is higher
(as in the case of N-hydroxy-piperidine) or lower (as in the case
of 1-hydroxy-6-nitro-benzotriazole), the catalytic activity is
diminished considerably.
Generally, amounts of from 0.1 to about 1 equivalent of N-hydroxy
compound are used, but smaller or larger amounts may also be
used.
Compounds suitable for catalysis are, inter alia,
1-hydroxy-benzotriazoles, for example
1-hydroxy-5,6-dimethylbenzotriazole,
1-hydroxy-5-methyl-benzotriazole, 1-hydroxy-6-methyl-benzotriazole,
1-hydroxy-5 -methoxy-benzotriazole,
1-hydroxy-4-methyl-benzotriazole, 1-hydroxy-6-bromo-benzotriazole,
1-hydroxy-6-chloro-benzotriazole, 1-hydroxy-5-chloro-benzotriazole;
cyclic hydroxamic acids, for example pyridones, such as
1-hydroxy-2-pyridone, 1-hydroxy-4-methyl-2-pyridone (cf. U.S. Pat.
No. 2 540 218), 1-hydroxy-4,6-dimethyl-2-pyridone,
1-hydroxy-3,4,6-trimethyl-2-pyridone, (cf. Belgian patent No. 738
288), 1-hydroxy-3,5-dichloro-4,6-dimethyl-2-pyridone,
N-hydroxy-succinimide or 1-hydroxy-2-oxo-2,3-dihydro-indoles, such
as 1-hydroxy-6-chloro-2-oxo-2,3-dihydro-indole [cf. J. Am. Chem.
Soc. 78, 222 (1956)]; cyclic hydroxamic acids containing a nitrogen
atom as a hetero atom in the ring, for example
3-hydroxy-4-oxo-3,4-dihydro-quinazoline,
3-hydroxy-2-methyl-4-oxo-3,4-dihydro-quinazoline,
3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine,
3-hydroxy-4-oxo-2-phenyl-3,4-dihydro-quinazoline (cf. J. Chem. Soc.
1960, pages 2157 - 2160); cyclic hydroxamic acids having an oxygen
or sulfur atom as a hetero atom in the ring, for example suitably
substituted 4-hydroxy-3,4-dihydro-3-oxo-1,4,2-benzoxazines and
4-hydroxy-3,4-dihydro-3-oxo-1,4,2-benzothiazines; or cyclic
thio-hydroxamic acids, for example
3-hydroxy-4-methyl-2,3-dihydro-thiazole-2-thione.
As substituents of the compounds of the formula I, there may be
mentioned alkyl or alkoxy groups having 1 or 2 carbon atoms, and
halogen atoms, especially chlorine.
The amino component of the formula III may either be employed in a
free form or as a salt with mineral acids or strong or weak organic
acids, for example of p-toluene-sulfonic acid, trifluoro-acetic
acid or acetic acid. If a salt is formed with a strong acid, the
amino group must be set free by adding a tertiary organic base,
such as triethylamine or N-ethyl-morpholine. It is also possible to
add a salt of these N-hydroxy compounds, such as the sodium salt of
1-hydroxy-benzotriazole. In this case, the amino group is set free
(formation of the sodium salt) at the same time that the
1-hydroxy-compound of the formula I, necessary for the catalysis,
is formed.
As protective groups V, the N-protective groups usual in peptide
chemistry (cf. Schroder-Lubke, The Peptides, New York and London
1965/66), preferably aralkyloxy-carbonyl groups, such as
benzyloxy-carbonyl, or tert.-alkyloxy-carbonyl groups, such as
tert.-butyloxy-carbonyl are used.
The OH-function in serine, threonine or tyrosine may be left
unprotected. It may, however, also be protected by suitable groups,
for example by tert.-butyl or benzyl groups.
For the manufacture of cysteine and cystine peptides, the
SH-function of cysteine has to be protected by the SH-protective
groups usual in peptide chemistry, or the reaction is carried out
on symmetrical or unsymmetrical cystine peptides which can then be
reduced, where required, to cysteine peptides.
The symbol W in the carboxyl component of the formula III stands
also for a polymer support which is linked to the carboxyl group
like an ester or an amide. Suitable polymers are polystyrene
cross-linked with 1 to 2% of divinyl-benzene, cross-linked
condensation products of phenol and formaldehyde, cross-linked
condensation products of phenol ether and formaldehyde, plastics
structures coated with chemically linked polystyrene, polybenzyl
resins or phenyl-substituted glass structures. Suitable linking
groups are, for example, halogeno-methyl, halogeno-acetyl or
amino-benzhydryl groups.
Condensation is advantageously performed in solvents, for example
dimethylformamide, dimethylacetamide, tetramethyl-urea or
phosphoric acid-tris-dimethylamide, optionally with an addition of
a small amount of water. The reaction temperature is advantageously
between 0.degree. and 40.degree.C, preferably room temperature.
The work-up is simple since the N-hydroxy compounds of the general
formula I, such as 1-hydroxy-benzotriazole or
3-hydroxy-4-oxo-3,4-dihydro-quinazoline, can be entirely removed by
shaking them with sodium or potassium bicarbonate or a sodium
carbonate solution. Other compounds used according to the
invention, for example 1-hydroxy-2-pyridone or
3-hydroxy-4-methyl-2,3-dihydro-thiazole-2-thione, are soluble in
water and can therefore be removed very easily. The N-hydroxy
compounds can also be extracted from sparingly soluble peptide
derivatives using isopropanol, alcohol, methanol, tetrahydrofuran
or hot water. The phenols formed during the reaction have solution
properties similar to those of N-hydroxy compounds and are
generally removed together with them.
Further purification is performed in usual manner: in the case of
lower peptides, for example, by distributing them between an
organic and an aqueous phase; by dissolving and precipitating them
or recrystallizing them from a suitable solvent; or by subjecting
them to chromatography, for example on "Sephadex LH-20" (Registered
Trademark) (cross-linked, partially etherified dextran gel), in an
organic solvent or an organic-aqueous mixture of solvents; or
optionally also by subjecting them to distribution chromatography
in only partially miscible solvent components.
In the case of higher peptides or proteins, it may be advantageous
to purify them only after the protective groups have been split
off, for example by means of equipotential precipitation,
crystallization, counter-current distribution, distribution
chromatography, gel chromatography or preparative
electrophoresis.
How to split off the protective groups depends on the nature of
these groups and has to take into consideration the stability of
the reaction products of the formula III or of the peptides on
which these compounds are based. The methods for splitting off the
groups are known in peptide chemistry (cf. Schroder-Lubke, loc.
cit.).
The addition of N-hydroxy compounds of the formula I does not
increase racemization in N-acyl peptide active esters, as is shown
by the racemization test known from Chem. Ber. 103, 788 (1970) (cf.
Table 2in the Experimental Part). In this racemization test, no
racemization could be observed either with nitrophenyl esters or
with trichlorophenyl esters with or without the addition of
N-hydroxy compounds. In contradistinction thereto,
pentachlorophenyl ester provided a tripeptide which had a
racemization degree of up to about 80%. By adding
1-hydroxy-benzotriazole, this high degree of racemization could be
reduced by 26 to 54%. When the sodium salt of 2-hydroxy-pyridine
was added, the inverse effect could be observed as already
mentioned: racemization was even intensified.
The addition of the N-hydroxy compounds of the formula I according
to the invention helps to overcome steric hindrance, hitherto an
unsurmountable obstacle to a number of reactions with activated
esters, for example to the reaction of Nps-isoleucine active esters
with H-Cys(Trt)-Ser-Leu-OH. The reaction of Nps-Ile-OTcp with
H-Cys(Trt)-Ser-Leu-OH in dimethyl-formamide could not be completed.
After 5 weeks, the reaction was discontinued since, in addition to
some end product and starting material, several decomposition
products could already be established by thin-layer chromatography.
When, however, for examaple 1-hydroxy-benzotriazole,
3-hydroxy-4-oxo-3,4-di-hydro-quinazoline or 1-hydroxy-2-pyridine
was added, the reaction was already complete after 15 to 20 hours
(cf. Table 3, Exper. Part).
The enormous catalytic power of these N-hydroxy compounds can also
be exploited in the solid-state peptide condensation method. For
example, according to J. Am. Chem. Soc. 90, 2953 (1966)
corresponding insoluble esters can be prepared from N-protected
amino acids and a poly-4-hydroxy-3-nitro-styrene resin and can then
be reacted with soluble amino compounds to yield the corresponding
amides or peptides. When the N-hydroxy compounds of the invention
are employed, the reaction time can be shortened considerably. Also
in the modified Merrifield-Solid State method, accordingg to which
N-protected amino acid active esters, for example nitrophenyl
ester, are often used, the additives of the invention increase the
yield and shorten the reaction time.
It is just the increase in yield which is critical in the
solid-state method for the success of such a synthesis. The
synthesis of a pentapeptide having a heavy steric hindrance and
belonging to the sequence of the insulin A chain demonstrates the
advantage of the additives of the invention (cf. Example 16). This
is the first time that a method has been found according to which
the steric hindrance, especially pronounced in the solid-state
synthesis, can be overcome.
The novel process is advantageous not only for minor peptides.
Major peptides, such as N.sup.(Al), N.sup.(B29) -bis-Boc-insulin
[cf. Hoppe-Seyler's Z. physiol. Chem. 352, 7 (1971)], a complicated
peptide synthesized from 51 amino acids, can be smoothly reacted,
within a few minutes, with N-acylamino acid active esters and
N-acyl peptide active esters to yield the corresponding N.sup.(B1)
-N-acyl-amino acid or N.sup.(B1) -N-acyl peptide-N.sup.(Al),
N.sup.(B29) -bis-Boc-insulins.
Compounds of the formula III may also be proteins, the free amino
groups of which react, in whole or in part, with the compounds of
formula II. Under extremely unfavorable solubility conditions, for
example aqueous dimethylformamide proves to be very suitable.
Appropriate proteins are, for example, gelatine which can be
partially degradated, serum albumin or casein.
After the protective groups have been split off, the reaction
products, for example those obtained from compounds of the formula
II with N.sup.(Al), N.sup.(B29) -bis-Boc-insulin, have a high
biological activity and modified physical and chemical as well as
immunological properties which are advantageous for the manufacture
of novel insulin preparations. Reaction products of compounds of
the general formula II with proteins may be employed, where
required after splitting-off of the protective groups, for the
production of antigens against the amino acids or peptides of
formula II from which these compounds are derived.
The peptides to be prepared according to the process of the
invention are synthesized from all amino acids to be found in
naturally occurring peptides in their L- or D-form. It is also
possible to use .beta.-amino acids, for example .beta.-alanine, or
other synthetic or semi-synthetic amino acids, for example
.alpha.-methyl-alanine, .alpha.-methyl-3,4-dioxy-L-phenyl-alanine
or .beta.-chloro-alanine.
The process of the invention is particularly advantageous since the
long reaction periods, which were hitherto required in many cases,
are now reduced to a few minutes. This is a special advantage, for
example, for the nitrophenyl ester method which is one of the most
frequently applied condensation methods for peptides.
Especially in the production of peptides on an industrial scale,
the process of the invention means good progress since the
throughput per unit of time can be increased considerably.
Another special advantage of the process of the invention is that
the catalytic effect of the N-hydroxy compounds of the invention is
fully developed especially in the above-cited strongly polar
solvents, which are the only ones used for the synthesis of higher
peptides because of their good dissolving properties.
After the protective groups have been split off, the peptides
prepared according to the invention can be used as therapeutic
compositions or as intermediate products for the manufacture of
other therapeutically interesting peptides, for example oxytocin,
vasopressin, glucagon, secretin, ACTH, thyrocalciton, gastrin TRH,
LR.sup.H or insulin.
The amino acids mentioned in the Disclosure and Examples are
abbreviated according to international rules. In addition, the
following abbreviations are used: Boc tert.-butyloxy-carbonyl Z
benzyloxy-carbonyl Nps o-nitrophenyl-sulfenyl OBu.sup.t tert.-butyl
ester OMe methyl ester OBzl benzyl ester ONp p-nitro-phenyl ester
OTcp 2,4,5-trichlorophenyl ester OPcp pentachloro-phenyl ester OSu
N-hydroxy-succinimide ester Trt trityl Bu.sup.t tert.-butyl Mbh
4,4'-dimethoxy-benzhydryl CHA cyclohexylamine
The following Examples serve to illustrate the invention.
EXAMPLE 1
Establishing the half-life period in the synthesis of
Z-Val-cyclohexyl amide from equimolar amounts of Z-Val-ONp and
cyclo-hexyl amine with an addition of various N-hydroxy-compounds,
and the dependency of the half-life period on the pK-value of the
N-hydroxy compounds.
a. Definition of the half-life period
Solution I: 0.5 .mu. mol of cyclohexylamine, 0.5 .mu. mol of
p-nitro-phenol and 1.0 .mu. mol of N-hydroxy compound per ml of
dimethylformamide.
Solution II: 1.0 .mu. mol of p-nitrophenol per ml of
dimethylformamide.
Solution III: 1.0 .mu. mol of N-hydroxy compound per ml of
dimethylformamide.
The UV-curves (range: 330-300 nanometers) of the solutions I, II
and III were compared with each other in order to find a wave
length which is favorable for measuring p-nitrophenol. At this wave
length, the extinction is measured. 50% of the extinction of a
solution of 1 nmol of Z-Val-ONp and 1 nmol of triethylamine per ml
of solvent were added to the value obtained for solution I at the
wave length chosen above. The sum of these two extinctions
represents the extinction at which half the amount of Z-Val-ONp has
reacted with cyclohexylamine.
The UV-curves were measured by means of a "Beckmann TK 1A"
apparatus at 21.degree.C. The substances were dissolved in
dimethylformamide.
b. Measuring of the half-life period
For the kinetic measurement of the half-life period t 1/2, a
solution of 1 .mu. mol of Z-Val-ONp, 1 .mu. mol of cyclohexylamine
and 1 .mu. mol of N-hydroxy compound per ml of solvent was measured
at the wave length defined sub (a). The period during which the
extinction percentage defined sub (a) was reached corresponds to
the half-time period of the reaction.
c. Measuring of the pK-values in a 0.5-molar solution
(diethylene-glycol dimethyl ether/water at a ratio of 6 : 4) at
30.degree.C.
0.5 mmol of substance was dissolved in a mixture of 6 ml of
diethylene-glycol dimethyl ether, 2.5 ml of 0.1N NaOH and 1.5 ml of
water and the solution was heated to 30.degree.C in a thermostat.
The pH was measured potentiometrically by means of a single-rod
glass electrode. According to the approximation formula pH - pK =
log .alpha./1-.alpha., where .alpha. is concentration, pK
.congruent. pH for .alpha. = 0.5. Hence, the above-measured
pH-value is the pK-value of this compound.
d. Table 1:
Comparison of the half-life periods in the synthesis of
Z-Val-cyclohexyl amide from molar amounts of Z-Val-ONp and
cyclohexylamine with the pH-values of the N-hydroxy compounds
added.
__________________________________________________________________________
Compound pK t 1/2(min)
__________________________________________________________________________
without additive -- 112.0 N-hydroxypiperidine 5.9 125.0
1-hydroxy-4-methyl-6-isopropyl-2-pyridone 4.14 19.0
3-hydroxy-2-methyl-4-oxo-3,4-dihydro- 4.11 1.9 quinazoline
3-hydroxy-4-oxo-3.4-dihydro-quinazoline 4.10 about 0.25
1-hydroxy-3.4.6-trimethyl-2-pyridine 4.09 14.8
1-hydroxy-4.6-dimethyl-2-pyridone 4.09 5.9
1-hydroxy-4-methyl-2-pyridone 4.08 3.2 1-hydroxy-2-pyridone 4.08
1.3 3-hydroxy-4-methyl-2.3-dihydro-thiazole- 4.08 1.5 2-thione
1-hydroxy-3.5-dichloro-4.6-dimethyl-2- 4.05 4.0 pyridone
1-hydroxy-2-oxo-2.3-dihydro-6-chloro-indole 4.05 1.5 acetic acid
(for comparison) 4.05 60.0 N-hydroxysuccinimide 4.04 4.8
1-hydroxy-5.6-dimethyl-benzotriazole 4.02 2.1
1-hydroxy-5-methyl-benzotriazole 4.02 2.8
1-hydroxy-6-methoxy-benzotriazole 4.00 2.0
1-hydroxy-5-methoxy-benzotriazole 4.00 3.6
1-hydroxy-4-methyl-benzotriazole 4.00 3.0 1-hydroxy-benzotriazole
4.00 3.5 3-hydroxy-4-oxo-3.4-dihydro-1.2.3-benzo- triazine 4.00
18.0 1-hydroxy-6-bromo-benzotriazole 3.91 8.9
1-hydroxy-6-chloro-benzotriazole 3.90 10.6
1-hydroxy-5-chloro-benzotriazole 3.89 13.9
1-hydroxy-6-trifluoromethyl-benzotriazole 3.72 39.0
1-hydroxy-5.6-dichloro-benzotriazole 3.7 35.0
1-hydroxy-6-nitro-benzotriazole 3.51 very slow
__________________________________________________________________________
EXAMPLE 2
Racemization test by gas chromatography according to F. Weygand et
al. [cf. Chem. Ber. 99, 1451 - 1460 (1966); op. cit. 103, 788 - 798
(1970)].
I. PREPARATION OF THE STARTING PRODUCTS
(a) HBr.H--Phe--ONp
4.2 g of Z--Phe--ONp were suspended in 10 ml of glacial acetic
acid. 10 ml of saturated HBr/glaciaal acetic acid were added and
the mixture was stirred for 1 hour at room temperature.
Subsequently, the hydrobromide was precipitated with a large amount
of ether and the precipitate was thoroughly washed with ether.
Yield: 3.5 g, melting point: 216.degree.-219.degree.C.
b. HBr.H--Phe--OPcp
5.47 g of Z-Phe-OPcp were treated as sub I(a).
Yield: 4.1 g, m.p. 205.degree.C with decomposition.
c. HBr.H--Phe--OTcp
4.78 g of Z-Phe-OTcp were treated as sub I(a).
Yield: 4.2 g, m.p. 220.degree.C with decomposition.
d. Boc--Leu--Phe--ONp
2.49 g of Boc--Leu--OH.1 H.sub.2 O were dissolved in ethyl acetate.
The ethyl acetate was dried with sodium sulfate, concentrated and
the residue was dried under greatly reduced pressure. The residue
was dissolved in 20 ml of absolute tetrahydrofuran and the solution
was cooled to -10.degree.C. While stirring and cooling with ice,
1.2 ml of N-ethyl-morpholine and then a solution of 1.27 g of
isobutyl-chloroformiate in a small amount of cold tetrahydrofuran
were added dropwise. Stirring was continued for 10 minutes at
-10.degree.C, then 3.35 g of HBr.H--Phe--ONp were added and then a
solution of 1.2 ml of N-ethyl-morpholine in 5 ml of tetrahydrofuran
was slowly added dropwise. Stirring was continued for 1 hour at
0.degree.C and for another hour at room temperature, the
precipitate was suction-filtered, the filtrate was concentrated and
the residue was triturated with isopropanol. Yield: 3.77 g, m.p.
150-152.degree.C. After recrystallization from isopropanol:
Yield: 3.10 g, m.p. 152.degree.-153.degree.C; [.alpha.].sub.D =
-32.2.degree. (c = 1, in dimethylacetamide).
e. Boc--Leu--Phe--OPcp
According to I (d), the mixed anhydried was prepared from 1.97 g of
Boc--Leu--OH. 1 H.sub.2 O, 0.93 ml of N-ethyl-morpholine and 1.0 g
of isobutyl chloroformiate and reacted with 3.55 g of
HBr.H--Phe--OPcp and 0.93 ml of N-ethyl-morpholine. Yield: 3.35 g,
m.p. 145.degree.-147.degree.C. After recrystallization from
isopropanol: Yield: 2.11 g, m.p. 163.degree.-164.degree.C;
[.alpha.].sub.D = -29.1.degree. (c = 1, in dimethyl-acetamide).
f. Boc--Leu--Phe--OTcp
According to I (d), a mixed anhydride was prepared from 2.3 g of
Boc--Leu--OH.1 H.sub.2 O, 1.1 ml of N-ethyl-morpholine and 1.17 g
of isobutyl chloroformiate and reacted with 3.6 g of HBr.H-Phe-OTcp
and 1.1 ml of N-ethyl-morpholine. Yield: 3.7 g, m.p.
143.degree.-145.degree.C. After recrystallization from isopropanol:
Yield: 3.2 g, m.p. 150.degree. - 151.degree.C; [.alpha.].sub.D =
-48.4.degree. (c = 1, in dimethylacetamide).
II. General Instruction for the preparation of
Boc--Leu--Phe--Val--OBu.sup.t from Boc-Leu-Phe active esters
A catalyzing additive (sodium salt of 1-hydroxy-benzotriazole or
2-hydroxy-pyridine) and 0.5 mmol of the corresponding Boc-Leu-Phe
active ester (250 mg of Boc--Leu--Phe--ONp, 313 mg of
Boc-Leu-Phe-OPcp, 279 mg of Boc-Leu-Phe-OTcp) are added to a
solution of 105 mg (0.5 mmol) of HCl.H-Val-OBu.sup.t in 2.5 ml of
0.2-molar N-ethyl-morpholine/dimethylacetamide solution. The
mixture is allowed to stand for the time given in Table 2, diluted
with 50 ml of ethyl acetate, the ethyl acetate solution is shaken
with NaHCO.sub.3 -solution, KHSO.sub.4 -solution, NaHCO.sub.3
-solution and NaCl-solution, dried with sodium sulfate and
concentrated. The residue is subjected, in a manner analogous to
Chem. Ber. 103, 788 - 798 (1970) and op. cit. 99, 1451 - 1460
(1966), to partial hydrolysis, to trifluoroacetylation and to gas
chromatography. The results of the racemization test by gas
chromatography are compiled in Table 2.
III. Table 2
Racemization tests in the synthesis of
Boc--Leu--Phe--Val--OBu.sup.t from Boc-Leu-Phe- active esters and
H--Val--OBu.sup.t under the influence of acylating catalysts Active
catalyst mol-equivalent reaction % of D- ester of catalyst period
Phe-L-Val
__________________________________________________________________________
ONp -- -- 70 hours < 2% ONp sodium salt of 1.0 1 hour 3.7%
2-hydroxy- pyridine ONp 1-hydroxy- 1.0 5 min. <2% benzotriazole
ONp 1-hydroxy-benzo- 0.1 5 min. <2% triazole OTcp -- -- 70 hours
< 2% OTcp sodium salt of 2- -- 1 hour < 2% hydroxy-pyridine
OTcp 1-hydroxy-benzo- 1.0 5 min. <2% triazole OPcp -- -- 70
hours 40% OPcp 2-hydroxy-pyridine 1.0 1 hour 40% OPcp
2-hydroxy-benzo- 1.0 5 min. 27% triazole OTcp 3-hydroxy-4-oxo- 1.0
10 min. <2% 3,4-dihydro-quinazo- line OTcp 3-hydroxy-2-methyl-
1.0 10 min. <2% 4-oxo-3,4-dihydro- quinazoline OTcp
1-hydroxy-2-pyridone 1.0 10 min. <2%
__________________________________________________________________________
EXAMPLE 3
Synthesis of Nps-Ile-Cys(Trt)-Ser-Leu-OH
a. Preparation of Nps-Ile-active esters
Molar amounts of Nps-isoleucine and trichlorophenol or
N-hydroxy-succinimide were reacted at 0.degree.C in ethyl acetate
with a slight excess amount of dicyclohexyl-carbodiimide (DCC). The
mixture was stirred for 1 hour at 0.degree.C, then for 3 hours at
room temperature, the precipitate was suction-filtered and the
filtrate was concentrated. The residue was recrystallized from
isopropanol.
Nps-Ile-OTcp, m.p. 95.degree. - 97.degree.C
Nps-Ile-OSu, m.p. 105.degree. - 108.degree.C
b. Nps-Ile-Cys(Trt)-Ser-Leu-OH
2.9 g (5 mmols) of H-Cys(Trt)-Ser-Leu-OH.H.sub.2 O (prepared
according to Chem. Ber. 103 (1970), page 2039) and 815 mg (5 mmols)
of 1-hydroxy-5,6-dimethyl-benzotriazole were dissolved in 25 ml of
dimethylformamide. 2.32 g (5 mmols) of Nps-Ile-OTcp were added and
the mixture was allowed to stand for 15 hours at room temperature.
(After this time, the starting tripeptide could no longer be
established by thin-layer chromatography). Dimethylformamide was
distilled off in vacuo and the residue was triturated with sodium
bicarbonate solution. The precipitate was suction-filtered and the
filtrate was eliminated. The precipitate was dissolved in ethyl
acetate and shaken with KHSO.sub.4 -solution and water, dried with
sodium sulfate and concentrated. The residue was triturated with
ether. The product was amorphous. Yield: 3.6 g (82%);
[.alpha.].sub.D.sup.22 -23.5.degree. (c=1, in dimethyl-acetamide).
Thin-layer chromatographic following of the synthesis of
Nps-Ile-Cys(Trt)-Ser-Leu-OH from Nps-Ile-OTcp or Nps-Ile-OSu and
H-Cys(Trt)-Ser-Leu-OH in the presence of various N-hydroxy
compounds as catalysts
0.2 mmol of the N-hydroxy compound and 111 mg (0.22 mmol) of
Nps-Ile-OTcp or 89.2 mg (0.24 mmol) of Nps-Ile-OSu were added to a
solution of 116.3 mg (0.2 mmol) of H-Cys(Trt)-Ser-Leu-OH.H.sub.2 0
in 2 ml of dimethylformamide. At different intervals of time, a
drop of the reaction solution was applied to a thin-layer silica
gel plate [Merck, "Kieselgel F 254" (Registered Trademark)]. The
solution was eluted with a mixture of methylene chloride and
methanol in a ratio of 8 : 2 and the chromatogram was developed
with ninhydrin. The results of this test are compiled in Table
3.
d. Table 3:
Synthesis of Nps-Ile-Cys(Trt)-Ser-Leu-OH from Nps-Ile-OTcp or
Nps-Ile-OSu and H-Cys(Trt)-Ser-Leu-OH with the use of various
N-hydroxy compounds as catalysts Ester N-hydroxy compound end of
reaction (reaction period) ______________________________________
OSu -- discontinued after 5 weeks since reaction not yet complete
OSu 3-hydroxy-4-oxo-3,4-dihydro- do. quinazoline OTcp -- do. OTcp
1-hydroxybenzotriazole about 15 hours OTcp
3-hydroxy-4-oxo-3,4-dihydro- about 15 hours quinazoline OTcp
3-hydroxy-2-methyl-4-oxo-3,4- about 7 days dihydro-quinazoline OTcp
3-hydroxy-4-oxo-2-phenyl-3,4- about 7 days dihydro-quinazoline OTcp
1-hydroxy-5,6-dimethyl-benzo- about 20 hours triazole OTcp
1-hydroxy-2-pyridone about 20 hours OTcp
1-hydroxy-4-methyl-2-pyridone about 20 hours OTcp
1-hydroxy-2,4-dimethyl-2- about 4 days pyridone
______________________________________
EXAMPLE 4
Systhesis of Z-Phe-Val-OMe
a. Using 1 equivalent of 1-hydroxy-benzotriazole and Z-Phe-OPcp
2.75 g (5 mmols) of Z-Phe-OPcp were added to a solution of 0.92 g
(5.5 mmols) of HCl.H-Val-OMe, 0.72 ml of N-ethyl-morpholine and 657
mg (5 mmols) of 1-hydroxy-benzotriazole in 10 ml of
dimethylformamide. The mixture was stirred for 5 minutes,
concentrated under greatly reduced pressure, the residue was
distributed between ethyl acetate and 2N sodium carbonate solution,
shaken once with KHSO.sub.4 -solution, three times with 2N sodium
carbonate solution and once with water, dried with sodium sulfate,
concentrated and the residue was triturated with petroleum
ether.
Yield: 2.08 g, m.p. 99.degree. - 104.degree.C. After
recrystallization from ethyl acetate/petroleum ether: Yield: 1.7 g
(83%), m.p. 112.degree.-114.degree.C.
b. Using 0.1 equivalent of 1-hydroxy-benzotriazole and
Z-Phe-OTcp
2.4 g (5 mmols) of Z-Phe-OTcp were added to a solution of 0.92 g
(5.5 mmols) of HCl.H-Val-OMe, 0.72 ml of N-ethyl-morpholine and
67.5 mg (0.5 mmol) of 1-hydroxy-benzotriazole in 10 ml of
dimethylformamide. The mixture was stirred for 5 minutes and worked
up as sub (a). Yield: 1.66 g (81%), m.p.
112.degree.-114.degree.C.
c. Using 1 equivalent of 1-hydroxy-benzotriazole (sodium salt) and
Z-Phe-ONp
865 mg (5.5 mmols) of the sodium salt of 1-hydroxy-benzotriazole
and 2.1 g (5 mmols) of Z-Phe-ONp were added to a solution of 0.92 g
(5.5 mmols) of HCl.H-Val-OMe in 10 ml of di-methylformamide. The
mixture was stirred for 5 minutes and worked up as sub (a). Yield:
1.65 g (80%), m.p. 116.degree.C.
EXAMPLE 5
Synthesis of Z-Val-Val-OMe
865 mg (5.5 mmols) of the sodium salt of 1-hydroxy-benzotriazole
and 1.87 g (5 mmols) of Z-Val-ONp were added to a solution of 0.92
g (5.5 mmols) of HCl.H-Val-OMe in 10 ml of dimethylformamide. The
mixture was stirred for 5 minutes and worked up as in Example 4
(a). Yield: 1.66 g (91%), m.p. 108-110.degree.C.
EXAMPLE 6
Synthesis of Z-Leu-Glu(OBu.sup.t)-Asn(Mbh)-Tyr(Bu.sup.t)-OH
(Sequence of the insulin A chain)
I. z-leu-Glu(OBu.sup.t)-Asn(Mbh)-Tyr(Bu.sup. t)-OH
1.5 g of Z-Leu-OPcp (2.2 mmols) were added to a solution of 1.53 g
(2 mmols) of H-Glu(OBu.sup.t)-Asn(Mbh)-Tyr(Bu.sup.t)-OH and 270 mg
of 1-hydroxy-benzotriazole (2mmols) in 5 ml of dimethylformamide.
The mixture was allowed to stand for 10 minutes and concentrated
under greatly reduced pressure. The residue was triturated with 2N
sodium carbonate solution which was then decanted, and the residue
was triturated with water. The precipitate was suction-filtered and
the filtrate was distributed between a 2.5% KHSO.sub.4 -solution
and ethyl acetate. The ethyl acetate solution was dried and
concentrated. The residue was triturated with ether/petroleum ether
(1:1) and suction-filtered. Yield: 1.8 g (89%), m.p.
174.degree.-176.degree.C.
This compound was identical, according to the thin-layer
chromatogram, with a sample prepared with Z-Leu-OSu in an analogous
manner. The reaction period was 24 hours and the yield was 84%.
The same result was obtained using
a. 3-hydroxy-4-oxo-3,4-dihydro-quinazoline,
b. 1-hydroxy-5,6-dimethyl-benzotriazole or
c. 1-hydroxy-pyridone, instead of 1-hydroxy-benzotriazole used
above.
Ii. preparation of the starting substance
H-Glu(OBu.sup.t)-Asn(Mbh)-Tyr(Bu.sup.t)-OH
a. Z-Asn(Mbh)-Tyr(Bu.sup.t)-OH
100 g of Z-Asn(Mbh)-Tyr(Bu.sup.t)-OMe (0.138 mol) [Chem. Ber. 103,
2041 - 2051 (1970)] were suspended in a mixture of 770 ml of dioxan
and 200 ml of water. At 37.degree.C, 138 ml of 1N NaOH were slowly
added dropwise (thymolphthalein used as indicator). When the
reaction was complete, the solution was acidified with 300 ml of 2N
citric acid. The precipitated product was suction-filtered and
washed with water. It was dissolved and precipitated from
methanol/water. Yield: 88 g (90%), m.p. 212.degree.-213.degree.C,
[.alpha.].sub.D = +7.6.degree. (c = 1, in dimethylformamide).
b. H-Asn(Mbh)-Tyr(Bu.sup.t)-OH
Hydrogen was passed through a solution of 87.5 g (0.123 mol) of
Z-Asn(Mbh)-Tyr(Bu.sup.t)-OH in glacial acetic acid, to which a
small amount of Pd(OH).sub.2 /BaSO.sub.4 -catalyst had been added,
until no more carbon dioxide developed. The solution was
concentrated and the residue was thoroughly triturated with a
saturated sodium acetate solution. The precipitate was
suction-filtered, dried over P.sub.2 O.sub.5 and dissolved and
precipitated from tetrahydrofuran petroleum ether. Yield: 63.5 g
(90%), m.p. 207.degree.-209.degree.C; [.alpha.].sub.D =
+11.1.degree. (c = 1, in glacial acetic acid).
c. Z-Glu(OBu.sup.t)-Asn(Mbh)-Tyr(Bu.sup.t)-OH
47.4 g (0.109 mol) of Z-Glu(OBu.sup.t)-OSu were added at room
temperature to a solution of 63 g (0.109 mol) of H-Asn(Mbh)-Tyr
(Bu.sup.t)-OH in 250 ml of dimethylformamide. The mixture was
allowed to stand for 20 hours at room temperature. The substance
was then precipitated with water, dried over P.sub.2 O.sub.5 and
dissolved and precipitated from ethyl acetate petroleum ether.
Yield: 75.5 g (77%), m.p. 166.degree.-170.degree.C, [.alpha.].sub.D
= +1.8.degree. (c = 1, in dimethylformamide).
d. H-Glu(OBu.sup.t)-Asn(Mbh)-Tyr(Bu.sup.t)-OH
74.7 g (83.3 mmols) of Z-Glu(OBu.sup.t)-Asn(Mbh)-Tyr(Bu.sup.t)-OH
were dissolved in a mixture of 400 ml of glacial acetic acid and
400 ml of methanol and the solution was catalytically hydrogenated
and worked up as sub (b). For purification, the substance was
boiled once with water, suction-filtered and dried. By boiling it
with ethyl acetate, the substance could be further purified. Yield:
53.4 g (84%), m.p. 221.degree.-223.degree.C, [.alpha.].sub.D =
+33.3.degree. (c = 1, in glacial acetic acid).
EXAMPLE 7
Synthesis of Z-Gln-Gly-Leu-Val-NH.sub.2 (sequence of secretin)
a. Z-Leu-Val-NH.sub.2
8.91 g of 1-hydroxy-benzotriazole, 8.6 ml of N-ethyl-morpholine and
30.8 g (60 mmols) of Z-Leu-OPcp were added to a solution of 13 g of
HBr.H-Val-NH.sub.2 (66 mmols) in 180 ml of dimethylformamide. The
mixture was stirred for 10 minutes at room temperature and then
concentrated under greatly reduced pressure. The residue was
successively stirred with 2N sodium carbonate solution, water, 2.5%
KHSO.sub.4 -solution and water, the precipitate was
suction-filtered and dried at 50.degree.C over P.sub.2 O.sub.5.
Yield: 21.3 g (98%).
The same result was obtained using
a. 3-hydroxy-4-oxo-3,4-dihydro-quinazoline,
b. 1-hydroxy-5,6-dimethyl-benzotriazole or
c. 1-hydroxy-pyridone, instead of 1-hydroxy-benzotriazole used
above.
b. HCl.H-Leu-Val-NH.sub.2
Pd(OH).sub.2 /BaSO.sub.4 -catalyst was added to a suspension of 21
g (57.8 mmols) of Z-Leu-Val-NH.sub.2 in a mixture of 200 ml of
dimethylformamide and 200 ml of methanol. While stirring, hydrogen
was passed through this reaction mixture while a pH-value of 5 was
maintained by adding dropwise a 1N methanolic HCl-solution by means
of an autotitrator. After no more HCl-solution was absorbed, the
catalyst was suction-filtered and the filtrate was concentrated.
The residue was triturated with ether, suction-filtered and dried
at 50.degree.C. Yield: 15.4 g (100%), m.p.
138.degree.-140.degree.C. The product was homogenous according to
the thin-layer chromatogram.
c. Z-Gly-Leu-Val-NH.sub.2
15.4 g (57.8 mmols) of HCl.H-Leu-Val-NH.sub.2, 7.52 ml of
N-ethyl-morpholine and 7.88 g of 1-hydroxy-benzotriazole were
dissolved in 120 ml of dimethylformamide. 22.5 g of Z-Gly-OTcp were
added, the mixture was allowed to stand for 5 minutes and then
concentrated under greatly reduced pressure. The residue was
triturated with 2N sodium carbonate solution, suction-filtered and
thoroughly washed with water. The substance was dried over P.sub.2
O.sub.5 and boiled with ethyl acetate. For purification, the
product could be dissolved and precipitated from
tetrahydrofuran/petroleum ether. Yield: 21.8 g (90%), m.p.
184.degree.-186.degree.C, [.alpha.].sub.D = -12.8.degree. (c = 1,
in dimethylacetamide).
d. HCl.H-Gly-Leu-Val-NH.sub.2
20.0 g. (47.6 mmols) of Z-Gly-Leu-Val-NH.sub.2 were catalytically
hydrogenated as sub (b) in 200 ml of a mixture of methanol and
dimethylformamide (1:1), and the product obtained was worked up as
sub (b). The substance was slightly hygroscopic and was dissolved
and precipitated from methanol/ether for purification. It was dried
over P.sub.2 O.sub.5 and paraffin scales.
Yield: 13.5 g (88%), m.p. 209.degree.-211.degree.C.
e. Z-Gln-Gly-Leu-Val-NH.sub.2
4 g (10 mmols) of Z-Gln-ONp were added to a solution of 3.23 g (10
mmols) of HCl.H-Gly-Leu-Val-NH.sub.2, 1.3 ml of N-ethyl-morpholine
and 1.35 g (10 mmols) of 1-hydroxy-benzotriazole in 20 ml of
dimethylformamide and the mixture was allowed to stand for 10
minutes. A jelly-like substance precipitated. It was diluted with
ether and the precipitated substance was suction-filtered. The
precipitate was dried for a short time and stirred with 2N sodium
carbonate solution. It was washed with water, KHSO.sub.4 -solution
and water, dried over P.sub.2 O.sub.5 and boiled with ethyl
acetate. It was allowed to cool to room temperature,
suction-filtered and washed with ethyl acetate and petroleum ether.
The product was dried at 40.degree.-50.degree.C over P.sub.2
O.sub.5 and paraffin scales. Yield: 5 g (91%), m.p.
242-244.degree.C. [.alpha.].sub.D.sup.22 = -25.2.degree. (c = 1, in
glacial acetic acid).
The same result was obtained using
a. 3-hydroxy-4-oxo-3,4-dihydro-quinazoline,
b. 1-hydroxy-5,6-dimethyl-benzotriazole or
1-hydroxy-pyridone, instead of 1-hydroxy-benzotriazole used
above.
EXAMPLE 8
Synthesis of Z-Pro-Ile-Gly-NH.sub.2
a. Z-Ile-Gly-NH.sub.2
44.4 g (0.1 mol) of Z-Ile-OTcp and 11 g (0.1 mole) of
HCl.H-Gly-NH.sub.2 as well as 1.6 g (10 mmols) of
3-hydroxy-4-oxo-3,4-dihydroquinazoline and 130 ml of
N-ethyl-morpholine were dissolved in 700 - 800 ml of
dimethylformamide. The solution was then concentrated under greatly
reduced pressure and the residue was triturated with a sodium
bicarbonate solution, the precipitate was suction-filtered, washed
with water, dried, dissolved in dimethylformamide and precipitated
with a mixture of ether/petroleum ether (1:1) for further
purification. Yield: 28.5 g, m.p. 202.degree.C, [.alpha.].sub.D =
+8.1.degree. (c = 1, in dimethylacetamide).
______________________________________ Analysis: C.sub.16 H.sub.23
N.sub.3 O.sub.4 (molecular weight: 321.4) calculated: C 59.80% H
7.21% N 13.08% found: 59.6 % 7. 2% 12. 8%
______________________________________
b. Z-Pro-Ile-Gly-NH.sub.2
A small amount of Pd(OH).sub.2 /BaSO.sub.4 -catalyst was added to a
solution of 20 g (62.2 mmols) of Z-Ile-Gly-NH.sub.2 in 600 ml of
methanol/dimethylformamide (3:1). While stirring, hydrogen was
passed through this reaction mixture and the pH-value was
maintained at 4.5 by adding dropwise 1N methanolic HCl-solution by
means of an autotitrator. After no more HCl-solution was absorbed,
the catalyst was suction-filtered and the filtrate was
concentrated. The residue was dissolved in 100 ml of
dimethylformamide. 23 g (62 mmols) of Z-Pro-ONp, 8.1 ml of
N-ethyl-morpholine and 1.5 g (9.4 mmols) of
3-hydroxy-4-oxo-3,4-dihydroquinazoline were added. The mixture was
stirred for 15 minutes and then concentrated under greatly reduced
pressure. The residue was dissolved in ethyl acetate and cooled. A
precipitate separated, it was suction-filtered and successively
triturated with a sodium bicarbonate solution, 2N HCl and water. It
was then dried and carefully triturated with ethyl acetate for
further purification. The precipitate was suction-filtered and
dried. Yield: 20.7 g (80%), m.p. 182.degree.-184.degree.C;
[.alpha.].sub.D = -63.2.degree. (c = 1, in methanol).
The same result was obtained using
a. 1-hydroxy-4-methyl-2-pyridone,
b. 1-hydroxy-6-methoxy-benzotriazole or
c. 1-hydroxy-5-methyl-benzotriazole, instead of the quinazoline
derivative used above.
EXAMPLE 9
Synthesis of Z-Asp(OBu.sup.
t)-Phe-Val-Gln-Trp-Leu-Ile-Asn-Thr-(Bu.sup.t )-OBu.sup.t
I. Preparation of the Starting substance CH.sub.3
COOH.H-Phe-Val-Gln-Trp-Leu-Ile-Asn-Thr(Bu.sup.t)-OBu.sup. t
a. Z-Asn-Thr(Bu.sup.t)-OBu.sup. t
42.85 g (161 mmols) of Z-Asn-OH, 43 g (161 mmols) of oily
HCl-H-Thr(Bu.sup.t)-OBu.sup. t (0btained by catalytic hydrogenation
of Z-Thr(Bu.sup.t)-OBu.sup. t with an addition of methanolic HCl at
a pH of 4.5) and 21.75 g (161 mmols) of 1-hydroxy-benzotriazole
were dissolved in 325 ml of dimethylformamide. 21 ml of
N-ethylmorpholine were added and a cold solution of 35.45 g of
dicyclohexyl-carbodiimide in 80 ml of dimethylformamide was added
at 0.degree.C. The mixture was stirred for 1 hour at 0.degree.C and
for another hour at room temperature, the precipitate was
suction-filtered and the filtrate was concentrated under greatly
reduced pressure. The residue was distributed between ethyl acetate
and NaHCO.sub.3 -solution. The organic phase was shaken with 2.5%
KHSO.sub.4 -solution, saturated NAHCO.sub.3 -solution and water,
dried with sodium sulfate and concentrated. The substance was
triturated with petroleum ether and suction-filtered, Yield: 65.8
g, m.p. 110.degree.-115.degree.C. Recrystallization from ethyl
acetate/petroleum ether. Yield: 52.4 g (68%), m.p.
120-122.degree.C, [.alpha.].sub.D.sup.20 = 7.6.degree. (c = 1, in
methanol).
b. HCl.H-Asn-Thr(Bu.sup.t)-OBu.sup. t
A small amount of Pd(OH).sub.2 /BaSO.sub.4 -catalyst was added to a
solution of 44.7 g (93.2 mmols) of Z-Asn-Thr(Bu.sup.t)-OBu.sup. t
in 300 ml of methanol. While stirring, hydrogen was passed through
this reaction mixture and the pH-value was maintained at 4.5 by
adding dropwise 1N methanolic HCl by means of an autotitrator.
After no more HCl-solution was absorbed, the catalyst was
suction-filtered and the filtrate was concentrated. The residue was
dissolved in ether, a small amount of insoluble material was
filtered off and the substance was placed in a refrigerator
overnight. Then the precipitate which had separated was
suction-filtered and washed with ether. Yield: 33.65 g (95.6%),
m.p. 110.degree.-113.degree.C, [.alpha.].sub.D.sup.20 = 2.6.degree.
(c = 1, in methanol).
______________________________________ Analysis: C.sub.16 H.sub.32
N.sub.3 O.sub.5 Cl molecular weight: 382 calculated: C 50.32% H
8.45% N 11.01% found: 50.0 % 9.0 % 10.9 %
______________________________________
c. Z-Ile-Asn-Thr(Bu.sup.t)-OBu.sup. t
A cold solution of 18.1 g of dicyclohexyl-carbodiimide in 70 ml of
dimethylformamide was added at 0.degree.C to a solution of 21.8 g
(82 mmols) of Z-Ile-OH, 31.3 g (82 mmols) of
HCl.H-Asn-Thr(Bu.sup.t)-OBu.sup. t, 11.1 g of
1-hydroxy-benzotriazole and 10.7 ml of N-ethyl-morpholine in 150 ml
of dimethylformamide. The mixture was stirred for 1 hour at
0.degree.C and for another hour at room temperature. The
precipitate was suction-filtered and the filtrate was concentrated.
The residue was worked up as sub (a). The resulting product was
dissolved in 150 ml of ethyl acetate, insoluble material was
separated by filtration and the residue was stirred into 750 ml of
petroleum ether. Yield: 42.37 g (87%), m.p.
144.degree.-146.degree.C, [.alpha.].sub.D.sup.20 = -20.5.degree. (c
= 1, in methanol).
______________________________________ Analysis: C.sub.30 H.sub.48
N.sub.4 O.sub.8 molecular weight: 592.75 calculated: C 60.82% H
8.15% N 9.46% found: 60.9 % 8.4 % 9.2 %
______________________________________
d. HCl.H-Ile-Asn-Thr(Bu.sup. t )-OBu.sup.t
38.6 g (65.2 mmols) of Z-Ile-Asn-Thr(Bu.sup.t )-OBu.sup.t were
catalytically hydrogenated in methanol as disclosed sub (b). The
substance was soluble in ether and provided an amorphous product
under greatly reduced pressure. Yield: 33.2 g (100%),
[.alpha.].sub.D.sup.30 = +5.9.degree. (c = 1, in methanol).
______________________________________ Analysis: C.sub.22 H.sub.43
ClN.sub.4 O.sub.6 molecular weight: 495.07 calculated: C 53.37% H
8.76% N 11.31% found: 53.0 % 9.0 % 10.9 %
______________________________________
e. Z-Leu-Ile-Asn-Thr(Bu.sup.t)-OBu.sup. t
A cold solution of 13.6 g of dicyclohexyl-carbodiimide in 60 ml of
dimethylformamide was added at 0.degree.C to a solution of 16.4 g
(61.8 mmols) of Z-Leu-OH, 30.6 g (61.8 mmols) of
HCl.H-Ile-Asn-Thr(Bu.sup. t)-OBu.sup. t, 8.34 g of
1-hydroxy-benzotriazole and 8.1 ml of N-ethyl-morpholine in 150 ml
of dimethylformamide. The mixture was stirred for 1 hour at
0.degree.C and for another hour at room temperature and the product
was worked up as disclosed sub (a). The substance was triturated
with petroleum ether and suction-filtered. Yield: 39.3 g, m.p.
173.degree.-176.degree.C. The substance was dissolved and
precipitated from ethyl acetate/petroleum ether. Yield 37.5 g
(86%), m.p. 173.degree.-176.degree.C, [.alpha.].sub.D.sup.30 =
-34.3.degree. (c = 1, in methanol).
______________________________________ Analysis: C.sub.36 H.sub.59
N.sub.5 O.sub.9 molecular weight: 705.9 calculated: C 61.26% H
8.42% N 9.92% found: 61.4 % 8.7 % 9.9 %
______________________________________
f. HCl.H-Leu-Ile-Asn-Thr(Bu.sup.t)-OBu.sup. t
35.2 g (50 mmols) of Z-Leu-Ile-Asn-Thr(Bu.sup.t)-OBu.sup. t were
catalytically hydrogenated in methanol according to (b). The
substance crystallized upon trituration with ether. Yield: 28.5 g
(94%), m.p. 192-193.degree.C with decomposition,
[.alpha.].sub.D.sup.30 = -17.9.degree. (c = 1, in methanol).
______________________________________ Analysis: C.sub.28 H.sub.54
ClN.sub.5 O.sub.7 molecular weight: 608.2 calculated: C 55.26% H
8.96% N 11.51% found: 55.3 % 9.0 % 11.4 %
______________________________________
g. Z-Trp-Leu-Ile-Asn-Thr(Bu.sup.t)-OBu.sup. t
A cold solution of 9.5 of dicyclohexyl-carbodiimide in 60 ml of
dimethylformamide was added at 0.degree.C to a solution of 26.2 g
(43.1 mmols) of HCl.H-Leu-Ile-Asn-Thr(Bu.sup.t)-OBu.sup.t, 14.6 g
(43.1 mmols) of Z-Trp-OH, 5.82 g of 1-hydroxy-benzotriazole and 5.6
ml of N-ethyl-morpholine in 150 ml of dimethylformamide. The
mixture was stirred for 1 hour at 0.degree.C and for another hour
at room temperature, the precipitate was suction-filtered and the
filtrate was concentrated. The residue was triturated with
NaHCO.sub.3 -solution and the precipitate was suction-filtered and
dried. Recyrstallization from isopropanol. Yield: 33.8 g (88%),
m.p. 228.degree.-229.degree.C, [.alpha.].sub.D.sup.30
=-39.5.degree. (c = 1, in methanol)
Analysis: C.sub.47 H.sub.69 N.sub.7 O.sub.10 molecular weight:
892.1 calculated: C 63.28% H 7.79% N 10.99% found: 63.0 % 7.8 %
10.7%
h. HCl.H-Trp-Leu-Ile-Asn-Thr(Bu.sup.t)-OBu.sup. t
31.2 g (35 mmols) of Z-Trp-Leu-Ile-Asn-Thr(Bu.sup.t)-OBu.sup.t t
were catalytically hydrogenated in 200 ml of
dimethylformamide/methanol (1:1) according to (b). The residue was
triturated with ether. Yield: 27.2 g (98%), m.p. 215.degree.C with
decomposition. [.alpha.].sub.D.sup.30 = -28.0.degree. (c = 1, in
methanol).
A sample was dissolved and precipitated from methanol/ether: m.p.
217.degree.C with decomposition, [.alpha.].sub.D.sup.30 =
-28.4.degree. (c=1, methanol)
Analysis: C.sub.39 H.sub.64 N.sub.7 O.sub.8 Cl molecular weight:
794.45 calculated: C 58.97% H 8.12% N 12.34% found: 57.2 % 8.2 %
11.8 %
i. Z-Phe-Val-OH
10.7 g of Z-Phe-Val-OMe were dissolved in 200 ml of dioxan/water (8
: 2). Titration is made against thymolphthalein using a total
amount of 27 ml of 1N NaOH. The solution was neutralized with 2N
HCl and concentrated. The residue was distributed between ethyl
acetate and 2N HCl. The ethyl acetate phase was washed with water,
dried with sodium sulfate and concentrated. The residue was
triturated with ether and suction-filtered.
Yield: 9.45 g (92%), M.p. 148.degree.-149.degree.C. The substance
was dissolved and precipitated from ethyl acetate:petroleum ether;
yield 9.2 g, m.p. 148.degree.-150.degree.C, [.alpha.].sub.D.sup.20
= -8.8.degree. (c=1, in methanol).
______________________________________ Analysis: C.sub.22 H.sub.26
N.sub.2 O.sub.5 molecular weight: 398.5 calculated: C 66.32% H
6.58% N 7.02% found: 66.6 % 6.7 % 7.2 %
______________________________________
k. Z-Phe-Val-Gln-OBu.sup. t
A cold solution of 4.2 g of dicyclohexyl-carbodiimide was added at
0.degree.C to a solution of 8.0 g (20 mmols) of Z-Phe-Val-OH, 4.8 g
of HCl.H-Gln-OBu.sup. t, 2.7 g of 1-hydroxy-benzotriazole and 2.6
ml of N-ethyl-morpholine in 40 ml of dimethylformamide. The mixture
was stirred for 1 hour at 0.degree.C and for another hour at room
temperature, the precipitate was suction-filtered and the filtrate
was concentrated. The residue was triturated with NaHCO.sub.3
-solution, suction-filtered and washed with water. Yield: 11.65 g
(100%), m.p. 219.degree.-221.degree.C with decomposition,
[.alpha.].sub.D.sup.20 = -35.7.degree. (c= 1, in methanol)
Analysis: C.sub.31 H.sub.42 N.sub.4 O.sub.7 molecular weight: 582.7
calculated: C 63.91% H 7.27% N 9.60% found: 64.2 % 7.7 % 9.7 %
1. Z-Phe-Val-Gln-OH
11.1 g (19 mmols) of Z-Phe-Val-Gln-OBu.sup. t were dissolved in 30
ml of trifluoroacetic acid. After 30 minutes at room temperature,
the solution was concentrated and the residue was triturated with
ether and suction-filtered. The product was boiled with ethyl
acetate for further purification, petroleum ether was added, the
precipitate was suction-filtered and dried. Yield: 8.45 g (85%)
m.p. 225.degree.-227.degree.C, [.alpha.].sub.D.sup.25 =
-10.7.degree. (c =1, in dimethylacetamide).
______________________________________ Analysis: C.sub.27 H.sub.34
N.sub.4 O.sub.7 molecular weight: 526.6 calculated: C 61.59% H
6.51% N 10.64% found: 61.6 % 6.7 % 10.4 %
______________________________________
m. Z-Phe-Val-Gln-Trp-Leu-Ile-Asn-Thr(Bu.sup.t)-OBu.sup. t
1.3 ml of N-ethyl-morpholine, 1.35 g of 1-hydroxy-benzotriazole
and, while stirring at 0.degree.C, a cold solution of 2.2 g of
dicyclohexyl carbodiimide in 20 ml of dimethylformamide were added
to a solution of 7.95 g (10 mmols) of
HCl.H-Trp-Leu-Ile-Asn-Thr(Bu.sup.t)-OBu.sup. t and 5.27 g (10
mmols) of Z-Phe-Val-Gln-OH in 100 ml of dimethylformamide. The
mixture was stirred for 1 hour at 0.degree.C and for another hour
at room temperature. The precipitate was suction-filtered and the
filtrate was concentrated. The residue was triturated with
NaHCO.sub.3 -solution and suction-filtered. The filter residue was
boiled with 1 l of ethanol. 5.6 g of a sparingly soluble residue
were obtained, m.p. 253.degree.-255.degree.C with decomposition,
[.alpha. ].sub.D.sup.30 = -15.3.degree. (c = 1, in
dimethylacetamide). Another 2.8 g crystallized from alcohol, m.p.
251.degree.-254.degree.C with decomposition. Total yield: 8.4 g
(66.5%).
______________________________________ Analysis: C.sub.66 H.sub.95
N.sub.11 O.sub.14 molecular weight: 1266.6 calculated: C 62.6% H
7.56% N 12.16% found: 62.2% 7.9 % 11.9 %
______________________________________
n. CH.sub.3
COOH.H.H-Phe-Val-Gln-Trp-Leu-Ile-Asn-Thr(Bu.sup.t)-OBu.sup.t
Hydrogen was passed through a solution of 7.6 g (6 mmols) of
Z-Phe-Val-Gln-Trp-Leu-Ile-Asn-Thr(Bu.sup.t)-OBu.sup.t) in 250 ml of
glacial acetic acid, to which a small amount of Pd(OH).sub.2
/BaSO.sub.4 -catalyst had been added, until no more carbon dioxide
developed. The catalyst was suction-filtered, the filtrate was
concentrated and the residue was triturated with ether. Yield: 7.4
g, m.p. 247.degree.-250.degree.C with decomposition. The product
was boiled with ethyl acetate and suction-filtered when cold.
Yield: 6.78 g (95%), m.p. 248.degree.-251.degree.C with
decomposition.
Ii.
z-asp(OBu.sup.t)-Phe-Val-Gln-Trp-Leu-Ile-Asn-Thr(Bu.sup.t)-OBu.sup.t
0.86 g (5.3 mmols) of 3-hydroxy-4-oxo-3,4-dihydroquinazoline and
2.66 g (5.3 mmols) of Z-Asp(OBu.sup.t)-OTcp were added to a
suspension of 6.32 g (5.3 mmols) of CH.sub.3
COOH.H-Phe-Val-Gln-Trp-Leu-Ile-Asn-Thr(Bu.sup.t)-OBu.sup.t in 150
ml of dimethylformamide. After having been stirred for 5 minutes,
the solution was concentrated and the residue was triturated with
2N sodium carbonate solution. The precipitate was suction-filtered
and washed with water. The filter residue was boiled with methanol.
A sparingly soluble residue was obtained: 5.3 g, m.p.
246.degree.-248.degree.C with decomposition; 0.5 g was obtained
from the filtrate, m.p. 244.degree.-246.degree.C with
decomposition; [.alpha.].sub.D.sup.25 = -15.0.degree. (c = 1, in
dimethylacetamide).
Total yeild: 5.8 g (76.4%)
Analysis: C.sub.74 H.sub.108 N.sub.12 O.sub.17 molecular weight:
1437.7 calculated: C 61.8% H 7.57% N 11.69% found: 61.1% 7.6 % 11.7
%
The same result was obtained using
a. 1-hydroxy-4-methyl-2-pyridone,
b. 1-hydroxy-6-methoxy-benzotriazole or
c. 1-hydroxy-5-methyl-benzotriazole, instead of the quinazoline
derivative mentioned above.
EXAMPLE 10
Synthesis of Z-Phe-Val-OMe
2 g of Z-phenyl-alanine-poly-4-hydroxy-3-nitrostyrene resin
containing 3 mmols of Z-phenyl-alanine and prepared according to J.
Am. Chem. Soc. 90, 2953 (1966), were suspended in 5 ml of
dimethylformamide and 162 mg (1 mmol) of
3-hydroxy-4-oxo-3,4-dihydroquinazoline, 168 mg (1 mmol) of
HCl.H-Val-OMe and 0.13 ml of N-ethyl-morpholine were added. The
mixture was stirred for 15 minutes, the precipitate was
suction-filtered, washed with dimethylformamide, the filtrate was
concentrated and the residue was distributed between ethyl acetate
and sodium bicarbonate solution. The ethyl acetate phase was washed
with 2N HCl, sodium bicarbonate solution and water, dried with
sodium sulfate and concentrated. The residue was triturated with
petroleum ether. The precipitate was suction-filtered and dried.
Yield: 390 mg (95%), m.p. 115.degree.C.
EXAMPLE 11:
Z-Tyr-Pro-insulin.sub.S -(31-63)-triaconta-tripeptide
40 mg (10 .mu.mols) of triaconta-tripeptide trifluoroacetate
prepared according to Z.Naturforsch. 24 b, 999 (1969) were reacted
in 1.8 ml of 90% dimethylformamide with 26 mg (60 mmols) of
Z-Tyr-ONp in the presence of 1.35 mg (10 .mu.mols) of
1-hydroxy-benzotriazole. After 15 minutes, the mixture was
precipitated with ether and the precipitate was suction-filtered.
After a washing operation with ether and methylene chloride, 36 mg
of a reaction product were obtained. According to the amine acid
analysis, the tyrosine content was 1.1 (calculated : 1).
R.sub.f :0.72 in a mixture of n-butanol/pyridine/acetic acid/water
(30:6:20:24), paper chromatography, ascending. R.sub.f of the
triaconta-peptide: 0.22.
EXAMPLE 12
N.sup.(Bl) -(Boc-Leu-Phe)-N.sup.(Al),N.sup.(B29)
-bis-Boc-insulin
16.7 mg (30 .mu.mols) of Boc-Leu-Phe-OTcp and 2.7 mg (20 .mu.mols)
of 1-hydroxy-benzotriazole were added in 2 ml of dimethylformamide
to 120 mg of Boc.sub.2 -insulin (20 .mu.mols), prepared according
to Hoppe-Seyler's Z.physiol. Chem. 352 7, (1971). The mixture was
stirred for 10 minutes at room temperature, precipitated with
ether. The precipitate was suction-filtered and washed with
ether.
Yield: 117 mg.
Amino acid analysis: Leu calculated: 7 found: 6.98 Phe calculated:
4 found: 3.96
Migration distance covered in paper electrophoresis at pH 2.2: 0.76
.times. insulin (Boc.sub.2 -insulin: 0.84 .times. insulin).
After the protective groups had been split off by a treatment with
trifluoracetic acid for 1 hour and precipitation with ether, the
compound covered the same distance in the paper electrophoresis as
insulin does. The reaction period was 3 hours without an addition
of 1-hydroxy-benzotriazole and partly brought about
cross-linking.
The same result was obtained using
a. 1-hydroxy-4-methyl-2-pyridone
b. 1-hydroxy-6-methoxy-benzotriazole or
c. 1-hydroxy-5-methyl-benzotriazole, instead of
1-hydroxybenzotriazole used above.
EXAMPLE 13
Boc-.beta.Ala-Tyr-Ser-Met-Glu(OBu.sup.t)-His-Phe-Arg-Trp-Cly-OH.4H.sub.2
0
5.36 g (4 mmols) of
H-Tyr-Ser-Met-Glu(OBu.sup.t)-His-Phe-Arg-Trp-Gly-OH.4H.sub.2 O,
prepared according to Liebig's Ann. Chem. 726, 183 (1969), were
reacted in 50 ml of dimethylformamide with 1.84 g (5 mmols) of
Boc-.beta.-Ala-OTcp in the presence of 540 mg (4 mmols) of
1-hydroxy-benzotriazole. After 15 minutes, 5.65 g (94%) of almost
chromatographically pure reaction product were precipitated with
ethyl acetate/ether (1:1) and recrystallized from 200 ml of 60%
methanol for complete purification. Yield: 4.92 g (81%). The
compound is identical with that obtained according to the
above-mentioned art without 1-hydroxy-benzotriazole, but the
reaction period was reduced from 20 hours 15 to
EXAMPLE 14:
N.sup.(.sup..alpha.1, .sup..epsilon.15, .sup..epsilon.26,
.sup..epsilon.41, .sup..epsilon.46)
-penta-(Leu-Phe)-trypsin-kallikrein inhibitor
90 mg of trypsin-kallikrein inhibitor from cattle's organs [cf.
Hoppe-Seyler's Z. physiol. Chem. 350, 1531 (1969)] were suspended
in 2 ml of dimethylformamide. 56 mg of Boc-Leu-Phe-OTcp and 13 mg
of 1-hydroxy-benzotriazole were added. Then water was added while
stirring (a total of 0.51 ml), until the whole was dissolved. The
solution was stirred for 15 minutes, concentrated under greatly
reduced pressure, and the residue was triturated with absolute
tetrahydrofuran. The precipitate was suction-filtered and dried.
Yield: 110 mg.
95.7 mg of the substance were dissolved in 2 ml of trifluoracetic
acid. The solution was allowed to stand for 30 minutes at room
temperature, concentrated and the residue was distributed between
water and ether, the aqueous phase was stirred with "Amberlite IR
45" (acetate form) and lyophilized.
______________________________________ Yield: 86 mg. Amino acid
analysis: Leu calculated: 7 found: 7.3 Phe calculated: 9 found: 9.2
______________________________________
EXAMPLE 15
Synthesis of H-Gly-Ile-Val-Glu-Gln-OH (insulin A 1-5)
I. preparation according to the solid-phase peptide synthesis
method using 3-hydroxy-4-oxo-3,4-dihydroquinazoline
a. Nps-Gln(Mbh)-polymer compound
A solution of 12.3 g of Nps-Gln(Mbh)-OH and 3.2 ml of
triethyl-amine in 60 ml of dimethylformamide was added to 20 g of a
polystyrene resin which had been chloromethylated according to
Merrifield, Biochemistry 3, 1358 (1964) and cross-linked with 2% of
divinyl-benzene, its chlorine content being 1.15 milli-equivalents
per gram. The mixture was shaken for 48 hours at 80.degree.C, the
precipitate was suction-filtered and the resin was washed several
times with dimethylformamide, ethanol and methanol. After drying it
in vacuo at 80.degree.C over P.sub.2 O.sub.5, the yield was 19.3 g.
The amino acid content of the acylamino acid - synthetic resin
compound was 1.15 mmols per gram.
Boc-Gly-Ile-Val-Glu(OBzl)-Gln(Mbh)-polymer compound
6 g of Nps-Gln(Mbh)-synthetic resin compound were subjected to the
following reaction cycle in a reactor provided with a glass frit
according to the Merrifield method (as above): b.sub.1 : Washing 3
times with 50 ml portions of glacial acetic acid.
b.sub.2 : Splitting off the N-protective groups by shaking for half
an hour with 1N solution of anhydrous HCl in glacial acetic acid,
filtering.
b.sub.3 : Washing with glacial acetic acid as disclosed sub
b.sub.1.
b.sub.4 : Washing three times with 50 ml portions of ethanol.
b.sub.5 : Washing three times with 50 ml portions of
dimethylformamide.
b.sub.6 : Neutralizing the hydrochloride grouping linked to the
released .beta.-amino groups of the resin by shaking for 10 minutes
with 50 ml of a 10% solution of triethylamine in
dimethylformamide.
b.sub.7 : Washing again with dimethylformamide as disclosed sub
b.sub.5.
b.sub.8 : Adding a dimethylformamide solution of 3.6 mmols of the
corresponding Boc-amino acid-trichlorophenyl ester in 40 ml of
dimethylformamide and 0.57 g (3.6 mmols) of
3-hydroxy-4-oxo-3,4-dihydroquinazoline and then shaking for 2
hours, finally filtering.
b.sub.9 : Washing with dimethylformamide as disclosed sub
b.sub.5.
b.sub.10 : Washing with absolute ethanol as disclosed sub
b.sub.4.
Each of the afore-mentioned washing operations took 3 minutes.
Subsequently, the solvent was removed by pressure filtration
(pressurized air: 0.3 atmospheres gauge) through the glass frit of
the reactor.
For linking an amino acid to the chain, the above-mentioned cycle
b.sub.1 to b.sub.10 was carried out once. For this, the following
Boc-amino-acid-2,4,5-trichlorophenyl esters were used successively
in step b.sub.8 of the disclosed cycle: Boc-Glu(OBzl)-OTcp,
Boc-Val-OTcp, Boc-Ile-OTcp and Boc-Gly-OTcp.
After drying over P.sub.2 O.sub.5 at 80.degree.C under greatly
reduced pressure, the yield of
Boc-Gly-Ile-Val-Glu(OBzl)-Gln(Mbh)-polymer compound was 5.3 g, the
peptide content being 8.2% (determined by acid hydrolysis and
quantitive determination of amino acids).
c. Release of the peptide
3 g of the acyl-peptide-polymer compound prepared according to (b)
were suspended in the above-mentioned reactor in 20 ml of anhydrous
trifluoracetic acid. Subsequently, a slow stream of anhydrous
hydrobromic acid was passed, within 90 minutes, with the exclusion
of moisture, from below through the glass frit of the reactor and
through the suspension lying above. The suspension was then
filtered and the resin was washed 3 times with 10 ml portions of
trifluoracetic acid. The combined trifluoracetic acid filtrates and
washing solutions were concentrated in vacuo. The residue was
digested twice while decanting with 50 ml portions of absolute
ether. After drying under greatly reduced pressure over P.sub.2
O.sub.5, the yield was 396 mg, m.p.: decomposition beginning at
225.degree.C, [.alpha.].sub.D.sup.22 = -51.4.degree. (c = 0.25, in
water). 114 mg of a colorless amorphous substance were obtained
from 200 mg of this crude product by distribution chromotography on
Sephadex LH 20 in a solvent system of butanol/acetic acid/water
(2:1:10). M.p. 240.degree.C with decomposition.
[.alpha.].sub.D.sup.22 = -71.4.degree. (c = 0.25, in water). Amino
acid analysis: see Table 4.
Ii. preparation according to the solid-phase peptide synthesis
method without using 3-hydroxy-4-oxo-3,4-dihydroquinazoline.
In this comparative test, the whole method of preparation was
qualitatively and quantitively identical with that disclosed sub I
without, however, using 3-hydroxy-4-oxo-3,4-dihydroquinazoline
according to step b.sub.8. In detail, the results of the
comparative test were the following: Yield of crude substance: 420
mg, m.p. decomposition beginning at 96.degree.C,
[.alpha.].sub.D.sup.22 = - 27.degree. (c = 0.25, in water). By
distribution chromatography on Sephadex LH 20, only 84 mg of a
product having the following physical data were obtained from 206
mg of crude product: M.p. decomposition beginning at 165.degree.C,
[.alpha.].sub.D.sup.22 -34.9.degree. (c = 0.25, in water). It was
not homogenous according to thin-layer chromatography. Amino acid
analysis: see Table 4.
Iii. preparation according to conventional methods
a. Boc-Gly-Ile-OH.CHA
26 ml of N-ethyl-morpholine and at 0.degree.C a solution of 44 g of
dicyclohexyl-carbodiimide in 50 ml of ice-cold dimethyl-formamide
were added to a solution of 35 g (0.2 mol) of Boc-Gly-OH, 38.4 g
(0.2 mol) of HCl.H-Ile-OMe and 27 g (0.2 mol) of
1-hydroxy-benzotriazole in 350 ml of dimethylformamide. The mixture
was stirred for 1 hour at 0.degree.C and for another hour at room
temperature, the precipitate was suction-filtered and the filtrate
was concentrated. The residue was distributed between ethyl acetate
and sodium bicarbonate solution. The ethyl acetate phase was shaken
with KHSO.sub.4 -solution, NaHCO.sub.3 -solution and water, dried
with sodium sulfate and concentrated. Yield:61.1 g in the form of
oil. The oil was dissolved in ethyl acetate and chromatographed by
means of a column packed with 185 g of basic Al.sub.2 O.sub.3
(Woelm, activity stage I). The ethyl acetate eluate was
concentrated. Yield: 55.8 g.
The above-mentioned oil was dissolved in 250 ml of a mixture of
dioxan and water (8:2) and a small amount of thymolphthalein was
added. Titration was then effected with a 1N sodium hydroxide
solution until the solution kept a blue color for a certain period
of time. Consumption: 179 ml of 1N NaOH. The solution was then
neutralized with KHSO.sub.4 -solution and the reaction mixture was
concentrated. The residue was dissolved in water. The solution was
covered with ethyl acetate and, while cooling with ice, it was
acidified with 2N H.sub.2 SO.sub.4 to reach a pH-value of 2-3 and
thoroughly stirred. The ethyl acetate phase was separated, washed
once with water, dried over sodium sulfate and concentrated. The
residue was dissolved in ether, and cyclohexylamine was added until
a basic reaction took place. The reaction mixture was placed in
ice, the precipitate was suction-filtered, washed with ether and
dried. Yield: 55.6 g (71.8%), m.p. 198.degree.-200.degree.C.
For further purification, the substance was dissolved in 100 ml of
methanol and the solution was stirred into 1.5ml of ether and
cooled. After 1 hour, the precipitate was suction-filtered. Yield:
45.5 g (59%), m.p. 204.degree.-205.degree.C, [.alpha.].sub.D.sup.22
= +17.4.degree. (c = 1, in methanol).
b. Z-Val-Glu(OBu.sup.t)-Gln(Mbh)-OH
65.2 g (81 mmols) of Z-Val-Glu(OBu.sup.t)-Gln(Mbh)-OMe prepared
according to Chem. Ber. 103, 2038 (1970), were suspended in about
400 ml of dioxan/water (8:2) and titrated with 1N NaOH against
thymolphthalein as an indicator. After the solution had kept its
blue color for a certain time, it was acidified with KHSO.sub.4
-solution and the precipitate was suction-filtered. The filter
material was boiled with acetone, the precipitate was
suction-filtered, dissolved and precipitated from
dimethylformamide/water.
______________________________________ Yield: 59 g (92%), m.p.
216-218.degree.C. Analysis: C.sub.42 H.sub.54 N.sub.4 O.sub.11
molecular weight: 790.9 calculated: C 63.72% H 6.85% N 7.05% found:
63.3 % 7.1 % 8.1 % ______________________________________
c. H-Val-Glu(OBu.sup.t)-Gln(Mbh)-OH
Hydrogen was passed through a suspension of 52 g (65.7 mmols) of
Z-Val-Glu(OBu.sup.t)-Gln(Mbh)-OH and a small amount of Pd(OH).sub.2
/BaSO.sub.4 -catalyst in 500 ml of glacial acetic acid until no
more CO.sub.2 escaped. The catalyst was then suction-filtered and
the filtrate was concentrated. The residue was triturated with
ether and suction-filtered. The precipitate was stirred for about 5
hours with a saturated sodium acetate solution, suction-filtered
and dried. The substance was finally boiled with methanol. Yield:
27 g (62.6%), m.p. 226-228.degree.C, [.alpha.].sub.D.sup.22 =
+9.0.degree. (c = 1, in glacial acetic acid).
______________________________________ Analysis: C.sub.34 H.sub.48
N.sub.4 O.sub.9 molecular weight: 656.8 calculated: C 62.20% H
7.35% N 8.53% found: 61.4 % 7.3 % 8.5 %
______________________________________
d. Boc-Gly-Ile-Val-Glu(OBu.sup.t)-Gln(Mbh)-OH
7.8 g (20 mmols) of Boc-Gly-Ile-OH.CHA were stirred at 0.degree.C
between 100 ml of ether and 40 ml of 2N citric acid. The ether
phase was separated, washed with water, dried with sodium sulfate
and concentrated. Yield: 4.6 g of oil.
2.09 g of 3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine and, at
0.degree.C, 2.76 g of dicyclohexyl-carbodiimide were added to a
solution of 3.9 g of the oily substance obtained (BocGly-Ile-OH) in
35 ml of dimethylacetamide. The mixture was stirred for 1 hour at
0.degree.C and for another hour at room temperature. The
precipitate which had separated was suctionfiltered and washed with
a small amount of dimethylacetamide. 5.85 g of finely ground
H-Val-Glu(OBu.sup.t)-Gln(Mbh)-OH were added to the filtrate and the
mixture was stirred for 5 hours at room temperature. A thick
precipitate separated. It was allowed to stand overnight and then
the substance was completely precipitated with 300 ml of water. The
precipitated substance was suction-filtered, washed with water and
boiled with 200 ml of ethanol. The solution was cooled to room
temperature and the precipitate was suction-filtered and carefully
washed with alcohol. Yield: 6.7 g (81.3%), m.p. 253-255.degree.C,
[.alpha.].sub.D.sup.22 = -2.1.degree. (c = 1, in
dimethylacetamide).
______________________________________ Analysis: C.sub.47 H.sub.70
N.sub.6 O.sub.13 molecular weight: 927.1 calculated: C 60.90% H
7.61% N 9.07% found: 61.0 % 7.6 % 9.2 %
______________________________________
e. H-Gly-Ile-Val-Glu-Gln-OH
200 mg of Boc-Gly-Ile-Val-Glu(OBu.sup.t)-Mbh-OH and 9.5 ml of
anisole were dissolved in 5 ml of trifluoracetic acid and the
solution was refluxed for 10 minutes. It was then concentrated and
the residue was triturated with ether. The precipitate was then
suction-filtered and dissolved in water. the aqueous solution was
stirred with "Amberlite IR 45" (acetate form) until a pH of 3 -4
was reached. The exchanger was suction-filtered and the filtrate
was clarified with charcoal. The clear aqueous solution was
freeze-dried. Yield: 50 mg [.alpha.].sub.D.sup.22 = -75.degree. (c=
0.25, in water). Substance was homogenous according to thin-layer
chromatogram. Amino acid analysis: see Table 4.
IV. TABLE 4
Comparison of the amino acid analyses and physical data of the
H-Gly-Ile-Val-Glu-Gln-OH prepared according to methods I, II and
III.
according to I according to II according to (solid-state
(solid-state III (convention- method using 3- method as in I, al
method) hydroxy-4-oxo- but without a 3,4-dihydro- catalyst)
quinazoline)
__________________________________________________________________________
[.alpha.].sup.22 (c=0.25,water) -71.5.degree. -39.4.degree.
-75.0.degree. decomposition from 240.degree.C from 165.degree.C
from 255.degree.C point thin-layer homogenous and 3 spots, one
homogenous chromatogram identical with thereof identi- III cal with
III Amino acid Glu 2.6 Glu 2.74 Glu 2.2 analysis Gly 1.0 Gly 1.0
Gly 1.0 (72 hours at Val 0.9 Val 0.445 Val 0.9 110.degree.C) Ile
0.87 Ile 0.33 Ile 0.9
__________________________________________________________________________
The results exhibited in Table 4 clearly demonstrate the
superiority of the process according to the invention even in the
solid-state method. According to the conventional method using
trichlorophenyl esters without the addition of the catalysts of the
invention, it has not been possible to obtain a material which is
even only approximately as homogenous as that obtained by this
invention.
Preparation of some catalysts
3-hydroxy-4-methyl-2,3-dihydro-thiazole-2-thione
a. 5-thioxo-6-oxa-4-thia-2-octanone
A solution of 185 g (2 mols) of chloroacetone in 300 ml of
methylene chloride was covered with 300 ml of water. At
10.degree.-20.degree.C, 320 g (2 mols) of C.sub.2 H.sub.5 O-CSSK
were introduced portionwise while stirring. Stirring was continued
for 2 hours at room temperature; the phases were separated, the
organic phase was washed once more with 100 ml of water, dried with
sodium sulfate and distilled. Boiling point 92.degree.-99.degree.C
under pressure of 0.05 mm mercury. Yield: 305 grams.
b. 5-thioxo-6-oxa-4-thia-2-octanone oxime
65.6 g of sodium acetate and 137 g of the
5-thioxo-6-oxa-4-thia-2-octanone obtained according to (a) were
added to a solution of 56.0 g of hydroxylamine hydrochloride in 400
ml of methanol. After stirring for 18 hours at room temperature,
the solvent was eliminated in vacuo, the residue was combined with
water, the mixture was extracted with methylene chloride, the
organic phase was dried with sodium sulfate and freed from the
solvent. The residue was dissolved in diisopropyl ether. By
cooling, a total amount of 101 g of oxime, m.p. 64.degree.C, could
be obtained from this solution in several fractions.
c. 3-hydroxy-4-methyl-2,3-dihydro-thiazole-2-thione
125.3 g of a solution of the oxime obtained according to (b) in 300
ml of methylene chloride were added dropwise, within 15 minutes,
while cooling and thoroughly stirring, to a solution of 104.4 g of
NaOH in 200 ml of water. Stirring was continued for 5 minutes, 500
ml of water were added, the phases were separated, the aqueous
phase was shaken twice with 100 ml portions of hexane and finally
acidified with concentrated hydrochloric acid to reach a pH-value
of 2. Stirring was continued for 1 hour in an ice bath, the
precipitate was carefully suction-filtered and thoroughly washed
twice with 300 ml of water each time. Yield: 61.5 g, m.p.
90.degree.C. Another 12 g of the same product could be obtained by
extraction of the aqueous solution with methylene chloride.
EXAMPLE 18
1-hydroxy-4,6-dimethyl-3,5-dichloro-2-pyridone
20 g of a 1-hydroxy-4,6-dimethyl-2-pyridone were dissolved in a
mixture of 75 ml of concentrated hydrochloric acid and 60 ml of
water and, at 10.degree. -15.degree.C, 21.6 g of chlorine were fed
in. Stirring was continued for 1 hour. The precipitate was
suction-filtered, washed with water and dried in vacuo. Yield: 22.9
g, m.p. 213.degree.C. Recrystallization from acetonitrile increased
the melting point to 216.degree.C.
* * * * *